Method for measuring hyaluronic acid

Abstract

A method for measuring hyaluronic acid in a biological sample which comprises (a) coating a solid support with hyaluronic acid; (b) incubating the sample with cartilage proteoglycan; (c) exposing the incubated sample to the coated solid support; (d) then exposing the coated solid support to a keratan sulfate-reactive antibody; (e) determining the amount of antibody linked to keratan sulfate; and (f) correlating the amount of antibody linked to the keratan sulfate to the amount of hyaluronic acid in the sample.

What is claimed is: 
     
       1. A method for measuring hyaluronic acid in a biological sample comprising: (a) coating hyaluronic acid to a solid support to produce a hyaluronic acid coated solid support;   (b) mixing the biological sample with cartilage proteoglycan, said cartilage proteoglycan having bound thereto keratan sulfate;   (c) exposing the mixture of Step (b) to the solid support of Step (a) to bind said proteoglycan to the hyaluronic acid on the solid support to produce a detectable product;   (d) exposing the product of Step (c) to a keratan sulfate specific antibody to bind to the keratan sulfate of the proteoglycan bound to the solid support;   (e) detecting the amount of said keratan specific antibody bound to the keratan sulfate in Step (d); and   (f) correlating the amount of said antibody detected in Step (e) to the hyaluronic acid in the biological sample,   wherein prior to Step (b) said cartilage proteoglycan is preincubated with a known amount of unbound hyaluronic acid and wherein said cartilage proteoglycan and said unbound hyaluronic acid are contacted with a solution comprising bovine serum albumin and polyoxyethylene sorbitan monolaurate.   
     
     
       2. A method according to claim 1 wherein said cartilage proteoglycan is present in an amount ranging from about 50 ng/0.2 ml total volume to about 500 ng/0.2 ml total volume. 
     
     
       3. A method for measuring hyaluronic acid in a sample comprising: (a) coating hyaluronic acid to a solid support to produce a hyaluronic acid coated solid support;   (b) mixing the sample with labelled cartilage proteoglycan;   (c) exposing the mixture of Step (b) to the solid support of Step (a) to bind said labelled proteoglycan to the hyaluronic acid on the solid support to produce a detectable product;   (d) determining the amount of product of Step (c) by detecting the labelled proteoglycan;   (e) correlating the amount of product in Step (d) to the amount of hyaluronic acid in the sample; and   (f) generating a standard curve for known amounts of hyaluronic acid by performing steps (a) through (e) on samples having known concentrations of hyaluronic acid and using the standard curve to convert the detected amount of product into a corresponding value for hyaluronic acid contained in the test sample,   wherein prior to Step (b) said cartilage proteoglycan is preincubated with a known amount of unbound hyaluronic acid and wherein said cartilage proteoglycan and said unbound hyaluronic acid are contacted with a solution comprising bovine serum albumin and polyoxyethylene sorbitan monolaurate.   
     
     
       4. A method according to claim 3 wherein said cartilage proteoglycan is present in an amount ranging from about 50 ng/0.2 ml total volume to about 500 ng/0.2 ml total volume. 
     
     
       5. A method for measuring hyaluronic acid in a biological sample comprising: (a) coating hyaluronic acid to a solid support to produce a hyaluronic acid coated solid support;   (b) mixing the biological sample with a first solution comprising bovine serum albumin and polyoxyethylene sorbitan monolaurate, cartilage proteoglycan and hyaluronic acid, said cartilage proteoglycan having bound thereto keratan sulfate;   (c) exposing the mixture of Step (b) to the solid support of Step (a) to bind said proteoglycan to the hyaluronic acid on the solid support to produce a detectable product;   (d) separating said detectable product from unreacted reagents;   (e) exposing said separated detectable product of Step (d) to a keratan sulfate specific monoclonal antibody to bind to the keratan sulfate of the proteoglycan bound to the solid support;   (f) detecting the amount of said keratan specific antibody bound to the keratan sulfate in Step (e); and   (g) correlating the amount of said antibody detected in step (f) to the hyaluronic acid in the biological sample.   
     
     
       6. A method according to claim 5 wherein said cartilage proteoglycan is present in Step (b) in an amount range from about 50 ng/0.2 ml total volume to about 500 ng/0.2 ml total volume.